US20040071686A1 - Treatment of alpha-galactosidase A deficiency - Google Patents

Treatment of alpha-galactosidase A deficiency Download PDF

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US20040071686A1
US20040071686A1 US10/423,225 US42322503A US2004071686A1 US 20040071686 A1 US20040071686 A1 US 20040071686A1 US 42322503 A US42322503 A US 42322503A US 2004071686 A1 US2004071686 A1 US 2004071686A1
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gal
preparation
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Douglas Treco
Kenneth Loveday
Marianne Borowski
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Shire Human Genetics Therapies Inc
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Transkaryotic Therapies Inc
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Priority to US10/423,225 priority Critical patent/US20040071686A1/en
Assigned to TRANSKARYOTIC THERAPIES, INC. reassignment TRANSKARYOTIC THERAPIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: TRECO, DOUGLAS A., BOROWSKI, MARIANNE, LOVEDAY, KENNETH
Publication of US20040071686A1 publication Critical patent/US20040071686A1/en
Priority to US11/403,618 priority patent/US7833742B2/en
Assigned to SHIRE HUMAN GENETIC THERAPIES, INC. reassignment SHIRE HUMAN GENETIC THERAPIES, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: TRANSKARYOTIC THERAPIES, INC.
Priority to US12/761,287 priority patent/US9267166B2/en
Priority to US14/995,540 priority patent/US9523113B2/en
Priority to US15/350,785 priority patent/US10245304B2/en
Priority to US16/276,039 priority patent/US11116823B2/en
Abandoned legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01022—Alpha-galactosidase (3.2.1.22)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters

Definitions

  • This invention relates to improved ⁇ -galactosidase A compositions for the treatment of ⁇ -galactosidase A deficiencies including Fabry disease.
  • Fabry disease is an X-linked inherited lysosomal storage disease characterized by severe renal impairment, angiokeratomas, and/or cardiovascular abnormalities, including ventricular enlargement and mitral valve insufficiency. Fabry disease also affects the peripheral nervous system, causing episodes of agonizing, burning pain in the extremities.
  • Fabry disease is caused by a deficiency in the enzyme ⁇ -galactosidase A ( ⁇ -Gal A).
  • ⁇ -Gal A The pathophysiology of Fabry Disease is well established: due to a lack of the lysosomal enzyme ⁇ -galactosidase A ( ⁇ -Gal A), there is accumulation of globotriaosylceramide (Gb 3 ) throughout the body.
  • the human enzyme has been expressed in Chinese Hamster Ovary (CHO) cells (Desnick et al., U.S. Pat. No. 5,356,804; Sicilnou et al., J Cell Biol. 119: 1137 (1992)); insect cells (Calhoun et al., WO 90/11353); and human cells (Selden et al., U.S. Pat. Nos. 6,083,725 and 6,458,574B1). Enzyme replacement therapy is a currently used method of treatment for Fabry disease.
  • ⁇ -Gal A dosing and administration strategies described herein reduce the amount and cost of ⁇ -Gal A required for ⁇ -Gal A replacement therapy and also reduce the required number of dose administrations.
  • the invention features a pharmaceutical composition that includes a human ⁇ -Galactosidase A ( ⁇ -Gal A)) preparation.
  • serum clearance of the ⁇ -Gal A preparation from the circulation is preferably less than 4 mL/min/kg on the linear portion of the AUC vs. close curve, more preferably less than about 3.5, 3, or 2.5 mL/min/kg, on the linear portion of the AUC vs. dose curve.
  • the exponent “b” is preferably at least 0.88, more preferably at least 0.90, and most preferably at least 0.92 or at least 0.94.
  • the ⁇ -Gal A is produced from human cells, e.g., primary human cells, e.g., primary human fibroblasts or a continuous human cell line.
  • the cells and/or the ⁇ -Gal A preparation isolated from the cells can be modified to provide an ⁇ -Gal A preparation with desirable glycosylation, phosphorylation or sialylation characteristics.
  • the ⁇ -Gal A is produced from non-human cells, e.g., CHO cells.
  • the cells and/or the ⁇ -Gal A preparation isolated from the cells can be modified to provide an ⁇ -Gal A preparation with desirable glycosylation, phosphorylation or sialylation characteristics.
  • the invention features a kit for the treatment of ⁇ -Gal A deficiency.
  • the kit includes (a) a human ⁇ -Gal A glycoprotein preparation, where at doses below serum or plasma clearance saturation levels, serum clearance of the ⁇ -Gal A preparation from the circulation is preferably less than 4 mL/min/kg on the linear portion of the area-under-the-curve (AUC) vs. dose curve, more preferably less than about 3.5, 3, or 2.5 mL/ml/kg, on the linear portion of the AUC vs. dose curve, and (b) instructions to administer the preparation to a subject in need thereof.
  • AUC area-under-the-curve
  • the kit can also include instructions to administer a unit dose of the ⁇ -Gal A preparation of between about 0.05 mg and 2.0 mg per kilogram of body weight of the subject (mg/kg).
  • the kit includes instructions to administer a unit dose of the ⁇ -Gal A preparation of between 0.05 and 2.0 mg/kg, preferably between about 0.05 and 1.0 mg/kg, more preferably between about 0.05 and 0.5 mg/kg, e.g., and between 0.05 and less than 0.3 mg/kg.
  • the unit dose is less than 0.3 mg/kg.
  • the kit can include instructions to administer a unit dose of the ⁇ -Gal A preparation of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg per kilogram of body weight.
  • the kit includes instructions to administer a unit dose of the ⁇ -Gal A preparation of between about 0.1 ⁇ 10 6 U/kg and 10 ⁇ 10 6 U/kg. In some embodiments, the kit includes instructions to administer a unit dose of the ⁇ -Gal A preparation of between 0.1 ⁇ 10 6 U/kg and 5 ⁇ 10 6 U/kg, preferably between about 0.1 ⁇ 10 6 U/kg and 3 ⁇ 10 6 U/kg. For example, the kit can include instructions to administer a unit dose of the ( ⁇ -Gal A preparation of about 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5 or up to 10 ⁇ 10 6 U/kg.
  • the kit also includes instructions to administer the unit close no more than once every 7 days.
  • the instructions can include instructions to administer the unit dose no more than once every 7 days, 10 days, 14 days, 21 days, 4 weeks, 6 weeks, 8 weeks or 10 weeks.
  • the invention features a kit for the treatment of ⁇ -Gal A deficiency.
  • the kit includes a human ⁇ -Gal A glycoprotein preparation and one or more of the following instructions: (a) instructions to administer the preparation to a subject in need thereof at a unit dose of between 0.05 and 2.0 mg/kg, preferably between about 0.05 and 1.0 mg/kg, more preferably between about 0.05 and 0.5 mg/kg, e.g., between 0.05 and less than 0.3 mg/kg; (b) instructions to administer a unit dose of the ⁇ -Gal A preparation of between 0.1 ⁇ 10 6 U/kg and 10 ⁇ 10 6 U/kg, e.g., between 0.1 ⁇ 10 6 U/kg and 5 ⁇ 10 6 U/kg, preferably between about 0.1 ⁇ 10 6 U/kg and 3 ⁇ 10 6 U/kg; or (c) instructions to administer the preparation no more than about once every 8 weeks, 6 weeks, 4 weeks, 21 days, 14 days, 10 days, or 7 days.
  • Kit 2 is comprised of the following major elements: packaging 4 , an ⁇ -Gal A preparation described herein 6 , and instructions 5 .
  • the kit may include an additional agent 10 .
  • the additional agent can be, e.g., a pharmaceutical buffer or solution, e.g., for dissolving or diluting the ⁇ -Gal A preparation 6 .
  • Instructions 8 can be, e.g., printed material on how to administer the preparation 6 and may include information on suitable dosage.
  • Preferred instructions comprise instructions to administer the ( ⁇ -Gal A preparation 6 in a unit dose described herein.
  • Packaging 4 is a box-like structure for holding a vial (or number of vials) containing an ⁇ -Gal A preparation of the invention 6 , instructions 8 , and, optionally, a vial (or number of vials) containing an agent 10 .
  • An individual skilled in the art can readily modify packaging 4 to suit individual needs.
  • the invention also features a method of selecting a unit dose range of ⁇ -Gal A for treatment of a subject having an ⁇ -Gal A deficiency.
  • the method includes: providing the body weight of a subject, e.g., weighing the subject or obtaining the subject's body weight from the subject, from a health care provider of the subject, or from a database; and determining the value of the range between 0.05 mg and 2 mg (e.g., between 0.05 and 0.5 mg or between 0.05 and less than 0.3 mg) of ( ⁇ -Gal A per kilogram of body weight of the subject.
  • the selected unit dose range can be used to select a regimen of ⁇ -Gal A replacement therapy for the subject.
  • the method can also include evaluating the subject for one or more of: basal ⁇ -Gal A levels, e.g., ⁇ -Gal A serum concentration; cardiovascular function; renal function; liver function, age, sex.
  • the unit dose saturates liver uptake of the ⁇ -Gal A by having C max (maximum serum concentration following drug infusion) greater than 2 ⁇ 10 ⁇ 9 M.
  • the invention also features a method of treating a subject having or at risk for having ( ⁇ -Gal A deficiency.
  • the method includes administering to a subject in need thereof a human ⁇ -Gal A glycoprotein preparation, where at doses below serum or plasma clearance saturation levels, serum clearance of the ⁇ -Gal A preparation following intravenous infusion from the circulation is preferably less than 4 mL/min/kg on the linear portion of the AUC vs. dose curve, more preferably less than about 3.5, 3, or 2.5 mL/min/kg, on the lineal portion of the AUC vs. dose curve, e.g., a human ( ⁇ -Gal A glycoprotein preparation described herein above.
  • the unit dose administered preferably saturates liver uptake of the ⁇ -Gal A.
  • the preparation is administered intravenously, although it may be formulated for oral, subcutaneous, or intrathecal administration, as described elsewhere herein.
  • the invention includes a method of treating a subject having or at risk for ⁇ -Gal A deficiency.
  • the method includes one or more of (a)-(c): (a) administering to a subject in need thereof a human ⁇ -Gal A glycoprotein preparation at a unit dose of between about 0.05 and 2.0 mg per kilogram of body weight, preferably between 0.05 and 1.0 mg/kg or between 0.05 and 0.5 mg/kg, e.g., between 0.05 and less than 0.3 mg/kg, e.g., about 0.25, 0.20, 0.15 or 0.1 mg per kilogram of body weight of the subject; (b) administering to a subject in need thereof a human ⁇ -Gal A glycoprotein preparation at a unit dose of the ( ⁇ -Gal A preparation of between 0.1 ⁇ 10 6 U/kg and 10 ⁇ 10 6 U/kg, e.g., between 0.1 ⁇ 10 6 U/kg and 5 ⁇ 10 6 U/kg, preferably between about 0.1 ⁇ 10 6 U/kg and
  • the human ⁇ -Gal A glycoprotein preparation is administered at least twice, preferably 3, 4, 5, 6 times or more, but no more than once every 7 days, preferably 10 days, more preferably 14 days or more, e.g., 21 days, 4 weeks, 6 weeks, 7 weeks, 8 weeks or more.
  • the unit dose saturates liver uptake of the ⁇ -Gal A, so as to allow administered ⁇ -Gal A to bypass the liver and be available to other tissues in the body.
  • the preparation is administered intravenously.
  • the invention features a unit dose of human ⁇ -Gal A described herein packaged in a container, e.g., a glass or plastic container or delivery device, e.g., a syringe.
  • the unit dose is equivalent to between 0.05 and 2 mg/kg, e.g., between 0.05 and 1.0 mg/kg, preferably between 0.05 and 0.5 mg/kg, more preferably between 0.05 and less than 0.3 mg/kg of body weight of the subject for which it is intended.
  • the activity of the ⁇ -Gal A preparation is generally between about 2.0 and 4.5 ⁇ 10 6 U/mg.
  • the container or delivery device can include between 2.0 and 32.0 mg of human ⁇ -Gal A described herein for an adult unit close, e.g., the container or delivery device can include about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25 or 30 mg of ⁇ -Gal A for an adult dose.
  • the ⁇ -Gal A preparations described herein can be predominantly cleared from the blood through mannose-6-phosphate (M6P) receptors. In preferred embodiments, less than 25%, 20%, 16%, 14% (as measured between 40 hours and 50 hours, e.g., approximately 44 hours, after dosing) or less of the ⁇ -Gal A preparation, e.g., a preparation described herein, is taken into the liver upon administration to a subject.
  • serum clearance of the ⁇ -Gal A preparation from the circulation is preferably less than 4 mL/min/kg on the linear portion of the AUC vs. dose curve, more preferably less than about 3.5, 3, or 2.5 mL/min/kg, oil the linear portion of the AUC vs. dose curve.
  • An ( ⁇ -Gal A preparation described herein exhibits a liver saturation curve as follows:
  • dose is the total dose (in mg) administered to a typical 75 kg patient.
  • the coefficient of variation (CV) can be, e.g., about 0.40. (Doses and amounts would be adjusted accordingly for larger or smaller patients).
  • the ⁇ -Gal A preparation of the compositions, methods and kits described herein is isolated from human cells genetically engineered to produce ⁇ -Gal A.
  • the ⁇ -Gal A preparation can be isolated from non-human cells (e.g., CHO cells), where the cell has been genetically engineered to produce ⁇ -Gal A.
  • one or more of: the ⁇ -Gal A expression construct, the human or non-human cells, or the ⁇ -Gal A isolated from the human or non-human cells can be modified to provide an ⁇ -Gal A preparation with altered glycosylation, e.g., altered glycan, sialylation or phosphate structures.
  • a non-human cell genetically engineered to produce a human ⁇ -Gal A (or the purified ⁇ -Gal A) can be modified to mimic the glycosylation characteristics of ⁇ -Gal A produced in human cells.
  • the cells can be modified, e.g., genetically engineered, to express one or more exogenous ⁇ -Gal A modifying enzyme, e.g., a glycosidase, glycosyl transferase, phosphoryl transferase, or sialyl transferase.
  • the ⁇ -Gal A coding sequence can be modified to have more or fewer (preferably more) glycosylation sites.
  • the cells can be exposed to one or more inhibitor or other modulator of glycosylation enzymes, e.g., kifunensine or swainsonine.
  • the ⁇ -Gal A once isolated from the cells, can be modified, e.g., cleaved or chemically modified (e.g. by changing the number of moles of sialic acid and/or mannose-6-phosphate per mole of ⁇ -Gal A), e.g., with a phosphatase inhibitor, kinase, glycosidase, glycosyl transferase, phosphoryl transferase, or sialyl transferase.
  • an ⁇ -Gal A preparation described herein is enriched in neutral, mono-sialylated and di-sialylated glycan structures (combined) relative to more highly sialylated structures such as tri-sialylated and tetra-sialylated structures.
  • a preferred ⁇ -Gal A preparation has one or more of: (a) at least about 22% neutral glycans, e.g., at least about 25% or 30% neutral glycans; (b) at least about 15%, 20%, or 25% mono-sialylated glycans; (c) at least about 35%, preferably at least about 40%, 45%, or 50% neutral and mono-sialylated glycans combined; (d) at least about 75%, 76%, 78% or more neutral, mono- and di-sialylated glycans combined; and (e) less than about 35%, preferably less than about 25%, 20%, 18% or about 15% tri- and tetra-sialylated glycan structures combined.
  • an ⁇ -Gal A preparation described herein has, on average, more than one complex glycan per monomer, preferably at least 50% complex glycans per monomer, e.g., an average of 1.5 complex glycans or more per monomer.
  • an ⁇ -Gal A preparation described herein has at least 5%, preferably at least 7%, 10% or 15% neutral glycans.
  • an ⁇ -Gal A preparation described herein has less than 45% phosphorylated glycans.
  • the preparation has less than about 35%, 30%, 25%, or 20% phosphorylated glycans.
  • an ⁇ -Gal A preparation described herein has a total proportion of sialylated glycans greater than about 45%, e.g., greater than 50% or 55%.
  • the ratio of sialic acid to mannose-6-phosphate in the ⁇ -Gal A preparation is greater than 1.5 to 1, preferably greater than 2 to 1, more preferably greater than 3 to 1, most preferably greater than 3.5 to 1 or higher.
  • the percent ratio of sialylated glycans to phosphorylated glycans is greater than 1, preferably greater than 1.5, more preferably greater than 2, e.g., greater than about 2.5 or 3.
  • the ⁇ -Gal A compositions and methods described herein are useful for treatment of individuals with ⁇ -Gal A deficiency.
  • the ⁇ -Gal A compositions and methods described herein provide treatments that are cost effective and minimize the required dosage and frequency of administrations of ⁇ -Gal A.
  • the invention features various methods of evaluating, e.g., analyzing, selecting or classifying an ⁇ -Gal A preparation, sample, batch or other composition.
  • the methods can be used to determine the structural and/or biological parameters (e.g., the carbohydrate composition, phosphate profile, sialylation profile, tissue distribution, or serum clearance characteristics) of the preparation or sample.
  • the methods are used to determine if the preparation or sample has one or more physical or functional property of an ⁇ -Gal A described herein.
  • a sample ⁇ -Gal A composition can be compared to a reference ⁇ -Gal A composition, e.g., a human ⁇ -Gal A composition described herein, e.g., a human ⁇ -Gal A having desirable pharmacokinetic or biological properties, such as a human ⁇ -Gal A prepared from human cells, e.g., human fibroblasts.
  • a reference ⁇ -Gal A composition e.g., a human ⁇ -Gal A composition described herein, e.g., a human ⁇ -Gal A having desirable pharmacokinetic or biological properties, such as a human ⁇ -Gal A prepared from human cells, e.g., human fibroblasts.
  • the methods are useful, inter alia, for quality control and/or bioequivalence studies of ⁇ -Gal A preparations.
  • the method includes obtaining or providing a test ⁇ -Gal A preparation and determining if the preparation has at least one (preferably at least two, more preferably at least three or more, e.g., at least four, five, six or seven) of the following structural characteristics: (1) is enriched in neutral, mono-sialylated and di-sialylated glycan structures (combined) relative to more highly sialylated structures, for example, has (i) at least about 22% neutral glycans, e.g., at least about 25% or 30% neutral glycans, (ii) at least about 15%, 20%, or 25% mono-sialylated glycans, (iii) at least about 35%, preferably at least about 40%, 45%, or 50% neutral and mono-sialylated glycans combined, and/or (iv) at least about 75%, 76%, 78% or more neutral, mono- and di-sialyl
  • the preparation is preferentially targeted to capillary/vascular endothelial cells, renal glomerular epithelial cells (podocytes) and glomerular mesangial cells, renal endothelial cells, cardiac myocytes, liver endothelial cells, liver sinusoidal cells, pulmonary cells, and/or neural cells; and (c) is not taken up by liver hepatocytes.
  • a test preparation that has one or more of the aforementioned characteristics can be selected, classified, formulated, packaged, or passed on to other downstream processing.
  • a preparation can be selected for a particular pharmaceutical use.
  • the possession by the test preparation of one or more (preferably at least two, more preferably at least three or more, e.g., at least four, five, six or seven) of the aforementioned structural parameters (1)-(7) is positively correlated with (and can thus be used to predict) desirable pharmacokinetic parameters or biological activity, e.g., one or more of the biological or pharmacokinetic characteristics (a-(c).
  • the correlation or predictive information can be used, e.g., to design an ⁇ -Gal A therapeutic preparation for a specific patient or a specific variant of Fabry disease (e.g., renal variant Fabry disease or cardiac variant Fabry disease).
  • the correlation or prediction information can be recorded (e.g., in a print or computer readable medium).
  • a biological activity or pharmacokinetic parameter of the test ⁇ -Gal A preparation or sample is predicted from its carbohydrate signature. In other embodiments, a biological activity or pharmacokinetic parameter of the preparation or sample is determined experimentally.
  • the result of the determination (which can be, e.g., a value for any of: the amount Of neutral, mono-, di-, tri- or tetra-sialylated glycans or combinations thereof; the amount of complex glycans; the amount of phosphorylated glycans; the amount of sialylated glycans; the ratio of sialic acid to mannose-6-phosphate on a mole per mole basis, or the ratio of sialylated glycans to phosphorylated glycans), is preferably entered into a record, e.g., a print or computer-readable record, such as a laboratory record or dataset.
  • a record e.g., a print or computer-readable record, such as a laboratory record or dataset.
  • the record can include other information, such as a specific sample identifier for the preparation, a date, an operator of the method, or information about the enzymatic activity, source, method of purification or biological activity of the preparation.
  • the record can be used to store or provide information about the test preparation.
  • the record can be used to provide information (e.g., to the government, a health care provider, insurance company or patient) related to the ⁇ -Gal A preparation or its use e.g., in the form of informational, marketing or instructional material, e.g., print material or computer readable material (e.g., a label).
  • the record or information derived from the record can be used, e.g., to identify the test preparation as suitable or unsuitable for pharmaceutical or therapeutic use.
  • a test ⁇ -Gal A preparation determined to have one or more of the aforementioned structural parameters (1)-(7) can be identified as having desirable pharmacokinetic parameters or biological activity (e.g., the aforementioned parameters (a)-(c).
  • the methods described herein can also be used to compare batch-to-batch variation of an ⁇ -Gal A preparation.
  • any of the structural or pharmacokinetic parameters described hereinabove can be evaluated for a plurality of ( ⁇ -Gal A batches, e.g., different batches made from the same purification protocol.
  • the method includes selecting a batch with less than a preselected range of variation (e.g., less than 10%, preferably less than 5%, more preferably less than 2.5% or less variation) from one or more of the aforementioned structural or biological parameters (1)-(7) or (a)-(c).
  • entering the result of the determinations into a record can include generating a dataset of the determinations, e.g., a print or computer-readable dataset.
  • the dataset can include a correlation of a determined structural characteristic with a predicted or experimentally evaluated biological activity or pharmacokinetic parameters.
  • the ⁇ -Gal A sample to be tested can be derived from any cell, but preferably is derived from a mammalian cell, e.g., a human or non-human cell, such as a CHO cell.
  • the carbohydrate signature of the sample has been modified, e.g., by art-recognized methods, before the determination step is performed, e.g., by glycoengineering, e.g., as described herein, by treatment with a glycosidation enzyme such as a glycosyl transferase or glycosidase, or treatment of the cell or preparation with a phosphoryl transferase, sialyl transferase, phosphatase inhibitor, kinase, or inhibitor of glycosylation, or by co-expression in the cell (e.g., via co-transfection) of a DNA encoding any of the foregoing enzymes or other carbohydrate modifying enzymes.
  • a glycosidation enzyme such as a
  • the carbohydrate signature of the sample can be obtained by methods known in the art, e.g., by ion exchange chromatography, high performance anion exchange (HPAE) chromatography, high performance liquid chromatography (HPLC), or mass spectroscopy. Evaluating the carbohydrate signature can include evaluating the composition, charge, phosphorylation, and/or sialylation of the glycans of the preparation.
  • HPAE high performance anion exchange
  • HPLC high performance liquid chromatography
  • the invention features a method of producing a human ⁇ -Gal A preparation (e.g., an improved ⁇ -Gal A preparation).
  • the method includes providing a human ⁇ -Gal A preparation harvested from a cell; and modifying the glycan structure of the ⁇ -Gal A preparation to match one or more (preferably at least two, more preferably at least three or more, e.g., at least four, five, six or seven) of the following parameters: (1) enrichment in neutral, mono-sialylated and di-sialylated glycan structures (combined) relative to more highly sialylated structures, for example, has (i) least about 22% neutral glycans, e.g., at least about 25% or 30% neutral glycans, (ii) at least about 15%, 20%, or 25% mono-sialylated glycans, (iii) at least about 35%, preferably at least about 40%, 45%, or 50% neutral and mono
  • the glycan structure can be modified by methods known in the art, e.g., by glycoengineering (e.g., by genetically engineering the cell to produce a human ⁇ -Gal A having a non-naturally occurring glycosylation site; and/or genetically engineering the cell to produce a glucosidase, glycosyl transferase, phosphoryl transferase, phosphatase, or sialyl transferase); by selective isolation of glycoforms during the ⁇ -Gal A purification process; by treatment of the cell or preparation with a carbohydrate modifying enzyme; or treatment of the cell or preparation with an inhibitor of glycosylation, e.g., kifunensine, and/or by co-expression in the cell (e.g., via co-transfection) of a DNA encoding any of the foregoing enzymes or other carbohydrate modifying enzymes.
  • glycoengineering e.g., by genetically engineering the cell to produce a human
  • the method includes the step of analyzing (e.g., assaying) one or more parameters of the carbohydrate signature, biological activity or pharmacokinetic parameter of the ⁇ -Gal A preparation after modification.
  • the invention also features a method of treating a subject, e.g., a human.
  • the method includes: providing or obtaining a panel of two or more ⁇ -Gal A preparations having different glycan characteristics; and selecting an ⁇ -Gal A preparation having a carbohydrate signature that matches one or more (preferably at least two, more preferably at least three or more, e.g., at least four, five, six or seven) of the aforementioned parameters (1)-(7) and/or (a)-(c) for treating the subject.
  • the method can also include administering one or more doses of a therapeutically effective amount of the selected ( ⁇ -Gal A preparation to the subject.
  • the subject can be evaluated before, during, and/or after the administration.
  • the tissue distribution or serum clearance of the ( ⁇ -Gal A preparation can be evaluated in the subject, e.g., evaluated repeatedly over time.
  • the dose of the preparation can then be adjusted according to the result of the evaluation.
  • the subject can also be evaluated or monitored for status, e.g., clinical status, in response to the administration of the ⁇ -Gal A preparation.
  • each parameter (1) to (7) or different combinations of parameters (1)-(7) can be correlated with having desirable pharmacokinetic or other biological properties for different populations, e.g., populations that differ by stage or type of disease (e.g., cardiac vs. renal type Fabry disease), age, gender, ethnic background, or genotype.
  • stage or type of disease e.g., cardiac vs. renal type Fabry disease
  • age e.g., gender, ethnic background, or genotype.
  • ⁇ -Gal A deficiency any deficiency in the amount or activity of this enzyme in a patient, resulting in abnormal accumulations of neutral glycolipids (e.g., globotriaosylceramide) primarily in capillary endothelial cells, renal glomerular epithelial cells (podocytes) and glomerular mesangial cells, and/or cardiac myocytes.
  • neutral glycolipids e.g., globotriaosylceramide
  • the deposits of this material can result in severe neuropathic pain (e.g., acroparasthesia and lacerative pain), serious renal and cardiovascular disease, and/or stroke.
  • the glycolipid accumulation may induce severe symptoms as typically observed in males who are suffering from Fabry disease.
  • the accumulation may induce relatively mild symptoms, as can sometimes be seen in some heterozygous female carriers of the defective gene.
  • Affected individuals have a greatly shortened life expectancy; death usually results from renal, cardiac, and/or cerebrovascular complications at approximately the fourth and fifth decade in life.
  • a “carbohydrate signature” of an ⁇ -Gal A preparation is one or more identifying characteristic of the glycan structure of a given preparation or sample of ( ⁇ -Gal A.
  • the carbohydrate signature can be qualitative or quantitative.
  • a carbohydrate signature of an ⁇ -Gal A preparation can include one or more of the following identifying characteristics: (a) the relative level, percentage range or specific value of complex vs.
  • the relative level, percentage range or specific value of neutral and sialylated e.g., mono-sialylated, di-sialylated, tri-sialylated and tetra-sialylated glycan structure
  • the relative level, percentage range or specific value of phosphorylated or non-phosphorylated glycans e.g., the relative level, percentage range or specific value of sialylated glycans
  • the relative or specific charge profile of the glycans of the preparation e.g., the ratio of sialic acid to mannose-6-phosphate; or the ratio of sialylated glycans to phosphorylated glycans.
  • FIG. 1 is a representation of the sequence of ⁇ -Gal A cDNA, including the sequence that encodes the signal peptide (SEQ ID NO:1).
  • FIG. 2 is a representation of the human ( ⁇ -Gal A amino acid sequence (SEQ ID NO:2).
  • FIG. 3A is a schematic map of pGA213C.
  • FIG. 3B is a diagrammatic representation of the targeting construct, pGA213C, and homologous recombination with the endogenous ⁇ -Gal A locus.
  • pGA213C is depicted as targeting sequences aligned above corresponding sequences on the X-chromosomal ⁇ -Gal A locus. Positions relative to the methionine initiation codon, ATG, are indicated by the numbers above the linear maps.
  • the activation unit containing murine dhfr, bacterial neo, and CMV promoter/aldolase intron sequences is shown above the position ( ⁇ 221) into which they were inserted by DNA cloning.
  • ⁇ -Gal A coding sequences are indicated by the darkened boxes.
  • ⁇ -Gal A non-coding genomic sequences are indicated by the lightly filled boxes. Large arrowheads indicate the direction of transcription for dhfr and ne
  • FIG. 4. is a chromatograph showing glycans released from ⁇ -Gal A made in human cells (ReplagalTM) vs ⁇ -Gal A made in CHO cells (FabrazymeTM). Both preparations were analyzed using HPAE-PAD on a Dionex BioLC Carbohydrate System. The glycan profiles indicate that there are significant differences in the glycan chains of ReplagalTM (top) and FabrazymeTM (bottom).
  • Fabrazyme is enriched in phosphorylated structures (peaks eluting at 65-69 minutes) and more highly sialylated structures (tetra-sialylated structures eluting at 56-60 minutes and tri-sialylated structures eluting at 51-55 minutes) as compared to ReplagalTM.
  • ReplagalTM is enriched in neutral (peaks at 33-36 minutes), mono-sialylated structures (peaks at 39-44 minutes) and di-sialylated structures (peaks at 45-49 minutes).
  • FIG. 5. is a graph showing internalization of ⁇ -Gal A made in human cells (ReplagalTM and ⁇ -Gal A made in CHO cells (FabrazymeTM) into cells.
  • Normal human fibroblasts were incubated in multi-well culture plates for 6 hours in the absence (control, not shown) or presence of ReplagalTM or FabrazymeTM.
  • This internalization is mannose-6-phosphate inhabitable, indicating that internalization is predominantly via mannose-6-phosphate receptors.
  • the results indicate that ReplagalTM and FabrazymeTM are not internalized comparably by the fibroblasts. FabrazymeTM is cleared more rapidly than ReplagalTM by mannose-6-phosphate receptor-mediated internalization.
  • FIG. 6 shows the molecular masses of ⁇ -Gal A made in human cells (ReplagalTM) (top) and ⁇ -Gal A made in CHO cells (FabrazyeTM) (bottom) as determined by MALDI-TOF mass spectroscopy.
  • the maximum of the major broad peak is at 50,755 and 50,705 Da, respectively, consistent with the expected molecular weight of the glycosylated monomer.
  • a leading shoulder at approximately 48,071 Da and 47,667 Da is present, representing the lower molecular weight glycoforms for ReplagalTM and FabrazymeTM, respectively.
  • the leading shoulder corresponding to the lower molecular weight glycoforms, is much more distinct in the spectrum of FabrazymeTM.
  • FIG. 7 is a charge profile of the glycans released from ( ⁇ -Gal A made in human cells (ReplagalTM) (bottom) and ⁇ -Gal A made in CHO cells (FabrazymieTM) (top). Glycans were derivatized with a fluorescent probe and compared by ion exchange chromatography on a GlycSepTM C column. The results show that ReplagalTM has a higher proportion of neutral and mono-charged glycans, and FabrazymeTM has a higher proportion of tri-charged glycans.
  • FIG. 8 is a chromatogram of FabrazymeTM (top) and ReplagalTM (bottom) as analyzed by reversed phase HPLC using a C4 reversed phase column (Vydac). Chromatograms obtained at 214 nm are shown. The leading shoulder, corresponding to the lower molecular weight glycoforms, is much more pronounced in FabrazymeTM.
  • FIG. 9 is a graph showing serum concentration (U/mil) of ⁇ -Gal A (ReplagalTM) made in human cells from Cynomolgus Monkey dosed IV at 1 mg/kg.
  • FIG. 10 is a graph showing dose proportionality of C max in animal models of ⁇ -Gal A (ReplagalTM) made in human cells.
  • FIG. 11 is a graph showing ReplagalTM plasma concentration (U/ml) following infusion at 0.2 mg/kg in a human subject.
  • FIG. 12 is a graph showing dose proportionality of C max in humans vs. monkey of ( ⁇ -Gal A (ReplagalTM) made in human cells.
  • FIG. 13 is a graph showing dose proportionality of area under the curve (AUC) in animal and humans for ( ⁇ -Gal A (ReplagalTM) made in human cells.
  • FIG. 14 is a graph showing liver distribution vs. dose in humans of an ⁇ -Gal A preparation (ReplagalTM) made in human cells.
  • FIG. 15 is a graph showing ReplagalTM plasma concentration (U/ml) following infusion at 0.2 mg/kg in a male and female human subject.
  • FIG. 16 is a schematic drawing of a kit containing an ⁇ -Gal A preparation described herein packaged in a vial and instructions for administering the preparation.
  • human ⁇ -Gal A can be made having modifications (e.g., in carbohydrate structure, e.g., glycan, phosphate or sialylation modifications) that result in a human ⁇ -Gal A preparation having pharmacokinetic properties that are desirable for enzyme replacement therapy for ⁇ -Gal A deficiency.
  • modifications e.g., in carbohydrate structure, e.g., glycan, phosphate or sialylation modifications
  • ⁇ -Gal A preparation as described herein, can be predominantly taken up by M6P receptors and has a serum clearance less rapid than that of human ⁇ -Gal A produced in non-human cells, e.g., CHO cells.
  • compositions and kits for treatment of ⁇ -Gal A deficiency described herein include such a ⁇ -Gal A preparations that are administered in a unit dose substantially smaller than what is currently used in the art.
  • the ⁇ -Gal A preparations described herein are administered in a unit dose of between 0.05 mg and 2.0 mg per kilogram of body weight (mg/kg), preferably between 0.05 and 5 mg/kg, more preferably between 0.05 and 0.3 mg/kg (e.g., about 0.1, 0.2, 0.25, 0.3, 0.4 or 0.5 mg/kg).
  • the unit dose can be, e.g., between 0.1 ⁇ 10 6 U/kg and 10 ⁇ 10 6 U/kg.
  • the unit dose of the ⁇ -Gal A preparation is between 0.1 ⁇ 10 6 U/kg and 5 ⁇ 10 6 U/kg, preferably between about 0.1 ⁇ 10 6 U/kg and 3 ⁇ 10 6 U/kg.
  • the ⁇ -Gal A preparations described herein are administered no more than once every 7 days, e.g., once every 10 days, 14 days or 21 days, or once every 4, 5, 6, 7 or 8 weeks. For some patients, even less frequent dosing may be possible, e.g., once every 9, 10, 11, 12 weeks or more.
  • the desirable pharmacokinetics result at least in part from the glycosylation patterns of the ⁇ -Gal A preparation.
  • the glycosylation patterns required for the desirable pharmacokinetics of human ⁇ -Gal A e.g., at least 50% complex glycans per ⁇ -Gal A monomer, on average; a ratio of sialic acid to mannose-6-phosphate (on a mole per mole basis) greater than 1.5 to 1, preferably greater than 2 to 1, more preferably greater than 3 to 1, most preferably greater than 3.5 to 1 or higher
  • ⁇ -Gal A e.g., at least 50% complex glycans per ⁇ -Gal A monomer, on average; a ratio of sialic acid to mannose-6-phosphate (on a mole per mole basis) greater than 1.5 to 1, preferably greater than 2 to 1, more preferably greater than 3 to 1, most preferably greater than 3.5 to 1 or higher
  • Certain representative embodiments are summarized and described in greater detail below.
  • the ⁇ -Gal A preparations described herein can be produced in any cell (an ⁇ -Gal A production cell) for the treatment of Fabry disease.
  • the compositions and methods described herein use human ⁇ -Gal A produced using standard genetic engineering techniques (based on introduction of the cloned ⁇ -Gal A gene or cDNA into a host cell), or gene activation, described in more detail below.
  • the human ⁇ -Gal A can be produced in human cells, which provide the carbohydrate modifications that are important for the enzyllle's pharmacokinetic activity.
  • human ⁇ -Gal A can also be produced in nonhuman cells, e.g., CHO cells. If the ⁇ -Gal A is produced in non-human cells, one or more of: the ⁇ -Gal A expression construct, the non-human cells, or the ⁇ -Gal A isolated from the non-human cells can be modified, e.g., as described herein below, to provide ⁇ -Gal A preparations having a glycosylation profile that results in desirable pharmacokinetic properties.
  • Purified human ⁇ -Gal A can be obtained from cultured cells, preferably genetically modified cells, e.g., genetically modified human cells or other mammalian cells, e.g., CHO cells. Insect cells can also be used.
  • genetically modified cells e.g., genetically modified human cells or other mammalian cells, e.g., CHO cells. Insect cells can also be used.
  • cells When cells are to be genetically modified for the purposes of treatment of Fabry disease, the cells may be modified by conventional genetic engineering methods or by gene activation.
  • a DNA molecule that contains an ⁇ -Gal A cDNA or genomic DNA sequence may be contained within an expression construct and transfected into primary, secondary, or immortalized cells by standard methods including, but not limited to, liposome-, polybrene-, or DEAE dextran-mediated transfection, electroporation, calcium phosphate precipitation, microinjection, or velocity driven microprojectiles (see, e.g., U.S. Pat. No. 6,048,729, incorporated herein by reference).
  • Viruses known to be useful for gene transfer include adenoviruses, adeno associated virus, herpes virus, mumps virus, polloviruis, retroviruses, Sindbis virus, and vaccinia virus such as canary pox virus.
  • the cells may be modified using a gene activation (“GA”) approach, for example, as described in U.S. Pat. No. 5,641,670; U.S. Pat. No. 5,733,761; U.S. Pat. No. 5,968,502; U.S. Pat. No. 6,200,778; U.S. Pat. No. 6,214,622; U.S. Pat. No. 6,063,630; U.S. Pat. No. 6,187,305; U.S. Pat. No. 6,270,989; and U.S. Pat. No. 6,242,218, each incorporated herein by reference.
  • GA-GAL Selden et al., U.S. Pat. Nos. 6,083,725 and 6,458,574B1.
  • the term “genetically modified,” as used herein in reference to cells, is meant to encompass cells that express a particular gene product following introduction of a DNA molecule encoding the gene product and/or including regulatory elements that control expression of a coding sequence for the gene product.
  • the DNA molecule may be introduced by gene targeting or homologous recombination, i.e., introduction of the DNA molecule at a particular genomic site. Homologous recombination may be used to replace the defective gene itself (the defective ( ⁇ -Gal A gene or a portion of it could be replaced in a Fabry disease patient's own cells with the whole gene or a portion thereof).
  • the term “primary cell” includes cells present in a suspension of cells isolated from a vertebrate tissue source (prior to their being plated, i.e., attached to a tissue culture substrate such as a dish or flask), cells present in an explant derived from tissue, both of the previous types of cells plated for the first time, and cell suspensions derived from these plated cells.
  • Secondary cells refers to cells at all subsequent steps in culturing. That is, the first time a plated primary cell is removed from the culture substrate and replated (passaged), it is referred to as a secondary cell, as are all cells in subsequent passages.
  • a “cell strain” consists of secondary cells which have been passaged one or more times; exhibit a finite number of mean population doublings in culture; exhibit the properties of contact-inhibited, anchorage dependent growth (except for cells propagated in suspension culture); and are not immortalized.
  • immortalized cell or “continuous cell line” is meant a cell from an established cell line that exhibits an apparently unlimited lifespan in culture.
  • Examples of primary or secondary cells include fibroblasts, epithelial cells including mammary and intestinal epithelial cells, endothelial cells, formed elements of the blood including lymphocytes and bone marrow cells, glial cells, hepatocytes, keratinocytes, muscle cells, neural cells, or the precursors of these cell types.
  • Examples of immortalized human cell lines useful in the present methods include, but are not limited to, Bowes Melanoma cells (ATCC Accession No. CRL 9607), Daudi cells (ATCC Accession No. CCL 213), HeLa cells and derivatives of HeLa cells (ATCC Accession Nos. CCL 2, CCL 2.1, and CCL 2.2), HL-60 cells (ATCC Accession No.
  • CCL 240 HT-1080 cells (ATCC Accession No. CCL 121), Jurkat cells (ATCC Accession No. TIB 152), KB carcinoma cells (ATCC Accession No. CCL 17), K-562 leukemia cells (ATCC Accession No. CCL 243), MCF-7 breast cancer cells (ATCC Accession No. BTH 22), MOLT-4 cells (ATCC Accession No. 1582), Namalwa cells (ATCC Accession No. CRL 1432), Raji cells (ATCC Accession No. CCL 86), RPMI 8226 cells (ATCC Accession No. CCL 155), U-937 cells (ATCC Accession No. CRL 15 93), WI-3 8VAI 3 sub line 2R4 cells (ATCC Accession No.
  • CLL 75.1 CCRF-CEM cells (ATCC Accession No. CCL 119), and 2780AD ovarian carcinoma cells (Van der Singh et al., Cancer Res. 48: 5927-5932, 1988), as well as heterohybridoma cells produced by fusion of human cells and cells of another species.
  • a clonal cell strain consisting essentially of a plurality of genetically identical cultured primary human cells or, where the cells are immortalized
  • a clonal cell line consisting essentially of a plurality of genetically identical immortalized human cells
  • the cells of the clonal cell strain or clonal cell line are fibroblasts.
  • the cells are secondary human fibroblasts, e.g., BRS-11 cells.
  • Example 1 provides additional guidance on the production of cells genetically engineered to produce human ( ⁇ -Gal A.
  • the cells are cultured under conditions permitting production and secretion of ⁇ -Gal A.
  • the protein is isolated from the cultured cells by collecting the medium in which the cells are grown, and/or lysing the cells to release their contents, and then applying protein purification techniques.
  • human ⁇ -Gal A can be made having modifications (e.g., carbohydrate, phosphate or sialylation modifications) that result in pharmacokinetic properties of the enzyme that are desirable for use in enzyme replacement therapy for ⁇ -Gal A deficiency.
  • modifications e.g., carbohydrate, phosphate or sialylation modifications
  • One method of making such human ⁇ -Gal A preparations is to produce human ⁇ -Gal A from human cells.
  • ⁇ -Gal A or indeed, of any glycoprotein
  • ⁇ -Gal A preparations described herein can also be produced from non-human cells, wherein either the cells, the ⁇ -Gal A coding sequence and/or the purified ⁇ -Gal A are modified.
  • non-human cells whose glycosylation machinery differs from human e.g., CHO cells
  • CHO cells can be genetically modified to express an enzyme of carbohydrate metabolism, e.g., ⁇ -2,6-sialyltransferase, that is present in human but not in CHO cells.
  • an enzyme of carbohydrate metabolism e.g., ⁇ -2,6-sialyltransferase
  • the cells can be genetically engineered to express an ⁇ -Gal A protein that has one or more modified glycosylation sites, e.g., a human or non-human cell can be genetically engineered to express an ⁇ -Gal A coding sequence in which one or more additional N-linked glycosylation sites have been added or deleted.
  • the additional glycosylation sites can be glycosylated by the cellular machinery in the cell, e.g., the CHO cell, in which the modified ⁇ -Gal A coding sequence is expressed, thus providing an ⁇ -Gal A preparation that has an increased circulatory half-life, cellular uptake, and/or improved targeting to heart, kidney or other appropriate tissues compared to the unmodified ⁇ -Gal A, e.g., when expressed in non-human cells.
  • ⁇ -Gal A can also be modified (e.g., after isolation from a genetically engineered non-human cell) to resemble human ( ⁇ -Gal A produced in human cells.
  • a human ⁇ -Gal A preparation isolated from a non-human cell can be modified, e.g., phosphorylated or cleaved (e.g., with neuraminidase or phosphatase) before administration to a subject.
  • the circulating half-life, cellular uptake and/or tissue targeting can also be modified, inter alia, by (i) modulating the phosphorylation of ⁇ -Gal A; (ii) modulating the sialic acid content of ⁇ -Gal A; and/or (iii) sequential removal of the sialic acid and terminal galactose residues, or removal of terminal galactose residues, on the oligosacharide chains on ⁇ -Gal A. Altered sialylation of ⁇ -Gal A preparations can enhance the circulatory half-life, cellular uptake and/or tissue targeting of exogenous ⁇ -Gal A.
  • a change in the ratio of moles of mannose-6-phosphate per mole of sialic acid per molecule of ⁇ -Gal A can also result in improved cellular-uptake, relative to that of hepatocytes, in non-hepatocytes
  • non-hepatocytes such as liver endothelial cells, liver sinusoidal cells, capillary/vascular endothelial cells, renal glomerular epithelial cells (podocytes) and glomerular mesangial cells, renal endothelial cells, pulmonary cells, renal cells, neural cells, and/or cardiac myocytes.
  • a preferred ratio of sialic acid to mannose-6-phosphate in the ⁇ -Gal A preparation is greater than 1.5 to 1, preferably greater than 2 to 1, more preferably greater than 3 to 1, most preferably greater than 3.5 to 1 or higher.
  • Glycoprotein modification e.g., when ⁇ -Gal A is produced in non-human cells
  • Glycoprotein modification can increase uptake of the enzyme in specific tissues other than liver and macrophages, e.g., increase uptake in capillary/vascular endothelial cells, renal glomerular epithelial cells (podocytes) and glomerular mesangial cells, renal endothelial cells, pulmonary cells, renal cells, neural cells, and/or cardiac myocytes.
  • glycoprotein modification methods human glycosylated ⁇ -Gal A preparations can be obtained, wherein between 35% and 85% of the oligosaccharides, preferably at least 50%, are charged.
  • Protein N-glycosylation functions by modifying appropriate asparagine residues of proteins with oligosaccharide structures, thus influencing their properties and bioactivities (Kukuruzinska & Lennon, Crit. Rev. Oral. Biol. Med. 9: 415-48 (1998)).
  • An ⁇ -Gal A preparation described herein can have a high percentage of the oligosaccharides being negatively charged, primarily by the addition of one to four sialic acid residues on complex glycans, or of one to two phosphate moieties on high-mannose glycans, or of a single phosphate and a single sialic acid on hybrid glycans.
  • Smaller amounts of sulfated complex glycans may also be present.
  • a high proportion of charged structures serves two main functions. First, capping of penultimate galactose residues by 2,3- or 2,6-linked sialic acid prevents premature removal from the circulation by the asialoglycoprotein receptor present on hepatocytes. This receptor recognizes glycoproteins with terminal galactose residues.
  • N-glycosylation is augmented by the fact that different asparagine residues within the same polypeptide may be modified with different oligosaccharide structures, and various proteins are distinguished from one another by the characteristics of their carbohydrate moieties.
  • a cell e.g., a non-human cell
  • a human ⁇ -Gal A having a non-naturally occurring glycosylation site
  • a human ⁇ -Gal A coding sequence to produce an ⁇ -Gal A protein having one or more additional glycosylation sites.
  • the additional glycosylation sites can be glycosylated (e.g., with complex glycans) by the cellular machinery in the cell, e.g., the CHO cell, in which the modified ⁇ -Gal A coding sequence is expressed, thus providing an ⁇ -Gal A preparation that has improved circulatory half-life, cellular uptake and/or tissue targeting compared to the unmodified ⁇ -Gal A, e.g., when expressed in non-human cells.
  • the cellular machinery in the cell e.g., the CHO cell
  • the modified ⁇ -Gal A coding sequence is expressed
  • the proportion of charged ⁇ -Gal A can be increased by selective isolation of glycoforms during the purification process.
  • the present invention provides for increasing the proportion of highly charged and higher molecular weight ⁇ -Gal A glycoforms by fractionation of ⁇ -Gal A species on chromatography column resins during and/or after the purification process.
  • the more highly charged glycoform species of ⁇ -Gal A contain more sialic acid and/or more phosphate, and the higher molecular weight glycoforms would also contain the fully glycosylated, most highly branched and highly charged species.
  • This fractionation process can occur on, but is not limited to, suitable chromatographic column resins utilized to purify or isolate ⁇ -Gal A.
  • suitable chromatographic column resins utilized to purify or isolate ⁇ -Gal A.
  • fractionation can occur on, but is not limited to, cation exchange resins (such as SP-SepharoseG), anion exchange resins (Q-SepharoseG), affinity resins (Heparin Sepharose-b, lectin columns) size exclusion columns (Superdex 200) and hydrophobic interaction columns (Butyl Sepharose) and other chromatographic column resins known in the art.
  • ⁇ -Gal A is produced in cells as a heterogeneous mixture of glycoforms which differ in molecular weight and charge, ⁇ -Gal A tends to elute in relatively broad peaks from the chromatography resins. Within these elutions, the glycoforms are distributed in a particular-manner depending on the nature of the resin being utilized. For example, on size exclusion chromatography, the largest glycoforms will tend to elute earlier on the elution profile than the smaller glycoforms.
  • the most negatively charged glycoforms will tend to bind to a positively charged resin (such as Q-SepharoseG) with higher affinity than the less negatively charged glycoforms, and will therefore tend to elute later in the elution profile.
  • these highly negatively charged glycoforms may bind less tightly to a negatively charged resin, such as SP Sepharose8, than less negatively charges species, or may not even bind at all.
  • Fractionation and selection of highly charged and/or higher molecular weight glycoforms of ⁇ -Gal A can be performed on any ⁇ -Gal A preparation, Such as that derived from genetically modified cells such as cells, e.g., human or non-human cells, modified by conventional genetic engineering methods or by gene activation (GA). It can be performed on cell lines grown in optimized systems to provide altered sialylation and phosphorylation as described herein, e.g., to provide a preparation with a ratio of sialic acid to mannose-6-phosphate (on a mole per mote basis) is greater than 1.5 to 1, preferably greater than 2 to 1, more preferably greater than 3 to 1, most preferably greater than 3.5 to 1 or higher.
  • a third approach for carbohydrate remodeling can involve modifying certain glycoforms on the purified ⁇ -Gal A by attachment of an additional terminal sugar residue using a purified glycosyl transferase and the appropriate nucleotide sugar donor.
  • This treatment affects only those glycoforms that have an appropriate free terminal sugar residue to act as an acceptor for the glycosyl transferase being used.
  • ⁇ 2,6-sialyltransferase adds sialic acid in an ⁇ -2,6-linkage onto a terminal Gal ⁇ 1,4GlcNAc-R acceptor, using CMP-sialic acid as the nucleotide sugar donor.
  • fucose ⁇ 1,3 transferases III, V and VI humans
  • galactose ⁇ 1,3 transferase porcine
  • galactose ⁇ 1,4 transferase bovine
  • mannose ⁇ 1,2 transferase yeast
  • sialic acid ⁇ 2,3 transferase rat
  • sialic acid ⁇ 2,6 transferase rat
  • glycosyl transferase can be removed from the reaction mixture by a glycosyl transferase specific affinity column consisting of the appropriate nucleotide bonded to a gel through a 6 carbon spacer by a pyrophosphate (GDP, UDP) or phosphate (CMP) linkage or by other chromatographic methods known in the art.
  • GDP pyrophosphate
  • CMP phosphate
  • the sialyl transferases is particularly useful for modification of enzymes, such as ⁇ -Gal A, for enzyme replacement therapy in human patients.
  • Use of either sialyl transferase with CMP-5-fluoresceinyl-neuraminic acid as the nucleotide sugar donor yields a fluorescently labeled glycoprotein whose uptake and tissue localization can be readily monitored.
  • a fourth approach for carbohydrate remodeling involves glyco-engineering, e.g., introduction of genes that affect glycosylation mechanisms of the cell, of the ⁇ -Gal
  • a production cell to modify post-translational processing in the Golgi apparatus is a preferred approach.
  • a fifth approach for carbohydrate remodeling involves treating ⁇ -Gal A with appropriate glycosidases to reduce the number of different glycoforms present. For example, sequential treatment of complex glycan chains with neuraminidase, ⁇ -galactosidase, and ⁇ -hexosaminidase cleaves the oligosaccharide to the trimannose core.
  • a sixth approach for glycan remodeling involves the use of inhibitors of glycosylation, e.g., kifunensine (an inhibitor of mannosidase I), swainsonine, or the like.
  • inhibitors of glycosylation e.g., kifunensine (an inhibitor of mannosidase I), swainsonine, or the like.
  • Such inhibitors can be added to the cultured cells expressing a human ⁇ -Gal A.
  • the inhibitors are taken up into the cells and inhibit glycosylation enzymes, such as glycosyl transferases and glycosidases, providing ⁇ -Gal A molecules with altered sugar structures.
  • glycosylation enzymes such as glycosyl transferases and glycosidases.
  • a seventh approach involves using glycosylation enzymes (e.g., glycosyl transferases or glycosidases) to remodel the carbohydrate structures in vitro, e.g., on an ⁇ -Gal A that has been isolated from a genetically engineered cell, as described herein.
  • glycosylation enzymes e.g., glycosyl transferases or glycosidases
  • Sialylation affects the circulatory half-life and biodistribution of proteins. Proteins with minimal or no sialic acid are readily internalized by the asialoglycoprotein receptor (Ashwell receptor) on hepatocytes by exposed galactose residues on the protein.
  • the circulating half-life of galactose-terminated ⁇ -Gal A can be altered by sequentially (1) removing sialic acid by contacting ⁇ -Gal A with neuraminidase (sialidase), thereby leaving the terminal galactose moieties exposed, and (2) removing the terminal galactoside residues by contacting the desialylated ⁇ -Gal A with ⁇ -galactosidase.
  • the resulting ⁇ -Gal A preparation has a reduced number of terminal sialic acid and/or terminal galactoside residues on the oligosaccharide chains compared to ⁇ -Gal A preparations not sequentially contacted with neuraminidase and ⁇ -galactosidase.
  • the circulating half-life of galactose-terminated ⁇ -Gal A can be enhanced by only removing the terminal galactoside residues by contacting the desialylated ⁇ -Gal A with ⁇ -galactosidase.
  • the resulting ⁇ -Gal A preparation has a reduced number of terminal galactoside residues on the oligosaccharide chains compared to ⁇ -Gal A preparations not contacted with ⁇ -galactosidase.
  • the resulting ⁇ -Gal A preparations are subsequently contacted with ⁇ -hexosaminidase, thereby cleaving the oligosaccharide to the trimannose core.
  • the sialic acid content of ⁇ -Gal A preparations can be increased by (i) isolation of the highly charged and/or higher molecular weight ⁇ -Gal A glycoforms during or after the purification process; (ii) adding sialic acid residues using cells genetically modified (either by conventional genetic engineering methods or gene activation) to express a sialyl transferase gene or cDNA; or (iii) fermentation or growth of cells expressing the enzyme in a low ammonium environment.
  • an ⁇ -Gal A preparation has less than 45% phosphorylated glycans.
  • the preparation has less than about 35%, 30%, 25%, or 20% phosphorylated glycans.
  • a desirable ratio of sialic acid:mannose-6-phosphate in the ⁇ -Gal A preparation is a ratio greater than 1.5 to 1, preferably greater than 2 to 1, more preferably greater than 3 to 1, most preferably greater than 3.5 to 1 or higher.
  • the phosphorylation of ⁇ -Gal A preparations can be modified, e.g., increased or decreased, by (i) adding or removing phosphate residues using cells genetically modified (either by conventional genetic engineering methods or gene activation) to express a phosphoryl transferase or phosphatase gene or cDNA; (ii) adding phosphatases, kinases, or their inhibitors to the cultured cells; or (iii) adding phosphatases, kinases, or their inhibitors to a purified ⁇ -Gal A preparation produced from a genetically engineered cell as described herein.
  • the second, N-acetylglucosamine-1-phosphodiester a-N-acetylglucosaminidase hydrolyzes the ⁇ -GlcNAc-phosphate bond exposing the Man-6-phosphate recognition site.
  • These enzymes can be induced or inhibited by methods known in the art to provide an ( ⁇ -Gal A preparation with desirable phosphorylation characteristics (e.g., with a desirable ration of sialylated to phosphorylated glycans).
  • an ⁇ -Gal A preparation with altered phosphorylation is obtained by first introducing into an ⁇ -Gal A production cell a polynucleotide which encodes for phosphoryl transferase, or by introducing a regulatory sequence by homologous recombination that regulates expression of an endogenous phosphoryl transferase gene.
  • the ⁇ -Gal A production cell is then cultured under culture conditions which results in expression of ⁇ -Gal A and phosphoryl transferase.
  • the ⁇ -Gal A preparation with increased phosphorylation compared to the ⁇ -Gal A produced in a cell without the polynucleotide is then isolated.
  • a glycosylated ⁇ -Gal A preparation with altered phosphorylation is obtained by adding a phosphatase inhibitor, e.g., bromotetramisole, or a kinase inhibitor, to cultured cells.
  • a phosphatase inhibitor e.g., bromotetramisole, or a kinase inhibitor
  • serum clearance of the ⁇ -Gal A preparation from the circulation is preferably less than 4 mL/min/kg on the linear portion of the AUC vs. dose curve, more preferably less than about 3.5, 3, or 2.5 mL/min/kg, on the linear portion of the AUC vs. dose curve.
  • the exponent “b” is preferably at least 0.88, more preferably at least 0.90, and most preferably at least 0.92, 0.94 or higher.
  • an ( ⁇ -Gal A preparation described herein is enriched in neutral, mono-sialylated and di-sialylated glycan structures (combined) relative to more highly sialylated structures such as tri-sialylated and tetra-sialylated structures.
  • a preferred ⁇ -Gal A preparation has one or more of: (a) at least about 22% neutral glycans, e.g., at least about 25% or 30% neutral glycans; (b) at least about 15%, 20%, or 25% mono-sialylated glycans; (c) at least about 35%, preferably at least about 40%, 45%, or 50% neutral and mono-sialylated glycans combined; (d) at least about 75%, 76%, 78% or more neutral, mono- and di-sialylated glycans combined; and (e) less than about 35%, preferably less than about 25%, 20%, 18% or about 15% tri- and tetra-sialylated glycan structures combined.
  • an ⁇ -Gal A preparation described herein has, on average, more than one complex glycan per monomer, preferably at least 50% complex glycans per monomer, e.g., 2 complex glycans or more per monomer.
  • an ⁇ -Gal A preparation described herein has at least 5%, preferably at least 7%, 10% or 15% neutral glycans.
  • an ⁇ -Gal A preparation described herein has less than 45% phosphorylated glycans.
  • the preparation has less than about 35%, 30%, 25%, or 20% phosphorylated glycans.
  • an the ⁇ -Gal A preparation described herein has a total proportion of sialylated glycans greater than about 45%, e.g., greater than 50% or 55%.
  • the ratio of sialic acid to mannose-6-phosphate in the ⁇ -Gal A preparation is greater than 1.5 to 1, preferably greater than 2 to 1, more preferably greater than 3 to 1, most preferably greater than 3.5 to 1 or higher.
  • the percent ratio of sialylated glycans to phosphorylated glycans is greater than 1, preferably greater than 1.5, more preferably greater than 2, e.g., greater than about 2.5 or 3.
  • the circulatory half-life of a human ⁇ -Gal A preparation is enhanced by complexing ⁇ -Gal A with polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the ⁇ -Gal A preparation is complexed using tresyl monomethoxy PEG (TMPEG) to form a PEGylated- ⁇ -Gal A.
  • TMPEG tresyl monomethoxy PEG
  • the PEGylated- ⁇ -Gal A is then purified to provide an isolated, PEGylated- ⁇ -Gal A preparation.
  • PEGylation of ⁇ -Gal A increases the circulating half-life, cellular uptake and/or tissue distribution of the protein.
  • ⁇ -Gal A may be purified to near-homogeneity from the cultured cell strains and/or conditioned medium of the cultured cell strains that have been stably transfected to produce the enzyme.
  • ⁇ -Gal A can be isolated from ⁇ -Gal A containing media using chromatographic steps. For example, 1 or more, e.g., 2, 3, 4, 5 or more chromatographic steps can be used.
  • the different steps of chromatography utilize various separation principles which take advantage of different physical properties of the enzyme to separate ⁇ -Gal A from contaminating material.
  • the steps can include: hydrophobic interaction chromatography on butyl Sepharose, ionic interaction on hydroxyapatite, anion exchange chromatography on Q Sepharose and size exclusion chromatography on Superdex 200.
  • Size exclusion chromatography can serve as an effective means to exchange the purified protein into a formulation-compatible buffer.
  • One purification process includes the use of butyl sepharose® chromatography as a first step in purification.
  • Other hydrophobic interaction resins such as Source Iso (Phamacia), Macro-Prep® Methyl Support (Bio-Rad), TSK Butyl (Tosohaas) or Phenyl Sepharose® (Pharmacia) can also be used.
  • the column can be equilibrated in a relatively high concentration of a salt, e.g., 1 M ammonium sulfate or 2 M sodium chloride, e.g., in a buffer of pH 5.6.
  • the sample to be purified can be prepared by adjusting the pH and salt concentration to those of the equilibration buffer.
  • the sample is applied to the column and the column is washed with equilibration buffer to remove unbound material.
  • the ⁇ -Gal A is eluted from the column with a lower ionic strength buffer, water, or organic solvent in water, e.g., 20% ethanol or 50% propylene glycol.
  • the ⁇ -Gal A can be made to flow through the column by using a lower concentration of salt in the equilibration buffer and in the sample or by using a different pH.
  • Other proteins may bind to the column, resulting in purification of the ⁇ -Gal A-containing sample which did not bind the column.
  • An alternative step of purification can use a cation exchange resin, e.g., SP Sepharose® 6 Fast Flow (Pharmacia), Source 30S (Pharmacia), CM Sepharose® Fast Flow (Pharmacia), Macro-Prep® CM Support (Bio-Rad) or Macro-Prep® High S Support (Bio-Rad), to purify ⁇ -Gal A.
  • a cation exchange resin e.g., SP Sepharose® 6 Fast Flow (Pharmacia), Source 30S (Pharmacia), CM Sepharose® Fast Flow (Pharmacia), Macro-Prep® CM Support (Bio-Rad) or Macro-Prep® High S Support (Bio-Rad)
  • the “first chromatography step” is the first application of a sample to a chromatography column (all steps associated with the preparation of the sample are excluded).
  • the ⁇ -Gal A can bind to the column at pH 4.4.
  • a buffer such as 10 mM sodium acetate, pH 4.4, 10 mM sodium citrate, pH 4.4, or other buffer with adequate buffering capacity at approximately pH 4.4, call be used to equilibrate the column.
  • the sample to be purified is adjusted to the ph and ionic strength of the equilibration buffer.
  • the sample is applied to the column and The column is washed after the load to remove unbound material.
  • a salt such as sodium chloride or potassium chloride, can be used to elute the ⁇ -Gal A from the column.
  • the ⁇ -Gal A can be eluted from the column with a buffer of higher pH or a combination of higher salt concentration and higher pH.
  • the ⁇ -Gal A can also be made to flow through the column during loading by increasing the salt concentration in the equilibration buffer and in the sample load, by running, the column at a higher pH, or by a combination of both increased salt and higher pH.
  • Another step of purification can use a Q Sepharose® 6 Fast Flow for the purification of ⁇ -Gal A.
  • Q Sepharose® 6 Fast Flow is a relatively strong anion exchange resin.
  • a weaker anion exchange resin such as DEAE Sepharose® Fast Flow (Pharmacia) or Macro-Prep® DEAB (Bio-Rad) can also be used to purify ⁇ -Gal A.
  • the column is equilibrated in a buffer, e.g., 10 mM sodium phosphate, pH 6.
  • the pH of the sample is adjusted to pH 6, and low ionic strength is obtained by dilution or diafiltration of the sample.
  • the sample is applied to the column under conditions that bind ⁇ -Gal A.
  • the column is washed with equilibration buffer to remove unbound material.
  • the ⁇ -Gal A is eluted with application of salt, e.g., sodium chloride or potassium chloride, or application of a lower pH buffer, or a combination of increased salt and lower pH.
  • the ⁇ -Gal A can also be made to flow through the column during loading by increasing the salt concentration in the load or by running the column at a lower pH, or by a combination of both increased salt and lower pH.
  • Another step of purification can use a Superdex® 200 (Pharmacia) size exclusion chromatography for purification of ⁇ -Gal A.
  • Other size exclusion chromatography resins such as Sephacryl® 200 HR or Bio-Gel® A-1.5 m can also be used to purify ⁇ -Gal A.
  • the preferred buffer for size exclusion chromatography is 25 mm sodium phosphate, pH 6.0, containing 0.15 M sodium chloride.
  • Other formulation-compatible buffers can also be used, e.g., 10 mM sodium or potassium citrate.
  • the pH of the buffer can be between pH 5 and pH 7 and should at contain a salt, e.g., sodium chloride or a mixture of sodium chloride and potassium chloride.
  • Another step of purification can use a chromatofocusing resin such as Polybuffer Exchanger PBE 94 (Pharmacia) to purify ⁇ -Gal A.
  • the column is equilibrated at relatively high pH (e.g., pH 7 or above), the pH of the sample to be purified is adjusted to the same pH, and the sample is applied to the column.
  • Proteins are eluted with a decreasing pH gradient to a pH such as pH 4, using a buffer system, e.g., Polybuffer 74 (Pharmacia), which had been adjusted to pH 4.
  • immunoaffinity chromatography can be used to purify ⁇ -Gal A.
  • An appropriate polyclonal or monoclonal antibody to ⁇ -Gal A (generated by immunization with ⁇ -Gal A or with a peptide derived from the ⁇ -Gal A sequence using standard techniques) can be immobilized on an activated coupling resin, e.g., NHS-activated Sepharose® 4 Fast Flow (Pharmacia) or CNBr-activated Sepharose® 4 Fast Flow (Pharmacia).
  • the sample to be purified can be applied to the immobilized antibody column at about pH 6 or pH 7. The column is washed to remove unbound material.
  • ⁇ -Gal A is eluted from the column with typical reagents utilized for affinity column elution such as low pH, e.g., pH 3, denaturant, e.g., guanidine HCl or thiocyanate, or organic solvent, e.g., 50% propylene glycol in a pH 6 buffer.
  • the purification procedure can also use a metal chelate affinity resin, e.g., Chelating Sepharose® Fast Flow (Pharmacia), to purify ⁇ -Gal A.
  • the column is pre-charged with metal ions, e.g., Cu +2 , Zn +2 , Ca +2 , Mg +2 or Cd +2 .
  • the sample to be purified is applied to the column at an appropriate pH, e.g., pH 6 to 7.5, and the column is washed to remove unbound proteins.
  • the bound proteins are eluted by competitive elution with imidazole or histidine or by lowering the pH using sodium citrate or sodium acetate to a pH less than 6, or by introducing chelating agents, such as EDTA or EGTA.
  • the ⁇ -Gal A preparations described herein exhibit a desirable circulatory half-life and tissue distribution, e.g., to capillary endothelial cells, renal glomerular epithelial cells (podocytes) and glomerular mesangial cells, and/or cardiac myocytes.
  • Such preparations can be administered in relatively low dosages.
  • the unit dose of administration can be between 0.05-2.0 mg per kilogram body weight (mg/kg).
  • the unit dose can be between 0.05 and 1.0 mg, between 0.5 and 0.5 mg/kg, or between 0.5 and 0.3 mg/kg.
  • Unit closes between 0.05 and 0.29 mg/kg are preferred, e.g., a unit dose of about 0.05, 0.1, 0.15, 0.2, 0.25, mg/kg. Assuming a specific activity of the ⁇ -Gal A preparation of between 2 and 4.5 ⁇ 10 6 U/mg, these values correspond to about 0.1 ⁇ 10 6 to 1.3 ⁇ 10 6 U/kg.
  • a preferred unit dose saturates liver uptake of the ⁇ -Gal A.
  • ⁇ -Gal A preparations described herein allow for the administration of the unit dose to a patient at relatively long intervals.
  • a unit dose can be administered no more than once every 7 days, 10 days, 14 days, 21 days, 4 weeks, 6 weeks, 8 weeks, 10 weeks or 12 weeks.
  • a preferred frequency of dosing is biweekly, monthly or bimonthly.
  • a patient can be monitored clinically to evaluate the status of his or her disease.
  • Clinical improvement measured by, for example, improvement in renal or cardiac function or patient's overall well-being (e.g., pain), and laboratory improvement measured by, for example, reductions in urine, plasma, or tissue Gb 3 levels, may be used to assess the patient's health status.
  • the frequency of ⁇ -Gal A administration may be reduced.
  • a patient receiving weekly injections of ⁇ -Gal A preparation may change to biweekly administration; a patient receiving biweekly injections of an ⁇ -Gal A preparation may switch to monthly administration; a patient receiving monthly injections of an ⁇ -Gal A preparation may switch to bimonthly injections.
  • the patient should be monitored for another period of time, e.g., several years, e.g., a three year period, in order to assess Fabry disease-related clinical and laboratory measures.
  • the administered dose does not change if a change in dosing frequency is made. This ensures that certain pharmacokinetic parameters (e.g.
  • maximal plasma concentration [C max ], time to maximal plasma concentration [t max ], plasma, half-life [t 1/2 ], and exposure as measured by area under the curve [AUC]) remain relatively constant following each administered dose. Maintenance of these pharmacokinetic parameters will result in relatively constant levels of receptor-mediated uptake of ⁇ -Gal A into tissues as dose frequencies change.
  • a patient is clinically evaluated between closes and a determination can be made upon evaluation as to the timing of the next dose.
  • Subcutaneous injections can be used to maintain longer term exposure to the drug.
  • Dosages of the ⁇ -Gal A preparations that are administered by intramuscular injections may be the same or different than those injected subcutaneously. In a preferred embodiment, intramuscular dosages are smaller and administered less frequently.
  • the ⁇ -Gal A preparation is preferably administered intravenously, e.g., in a intravenous bolus injection, in a slow push intravenous injection, or by continuous intravenous injection. Continuous IV infusion (e.g., over 2-6 hours) allows the maintenance of specific levels in the blood.
  • a patient with a typical variant of Fabry disease e.g., exhibiting, predominantly cardiovascular abnormalities or renal involvement
  • the dose is adjusted as needed.
  • a patient with the cardiac variant phenotype who is treated with ⁇ -Gal A enzyme replacement therapy will have a change in the composition of their heart and improved cardiac function following therapy. This change can be measured with standard echocardiography which is able to detect increased left ventricular wall thickness in patients with Fabry disease (Goldman et al., J Am Coll Cardiol 7:1157-1161 (1986)).
  • Serial echocardiographic measurements of left ventricular wall thickness can be conducted during therapy, and a decrease in ventricular wall size is indicative of a therapeutic response.
  • MRI cardiac magnetic resonance imaging
  • Fabry disease reveals deposited lipid within the myocardium compared with control patients (Matsui et al., Am Heart J 117: 472-474. (1989)).
  • Serial cardiac MRI evaluations in a patient undergoing enzyme replacement therapy can reveal a change in the lipid deposition within a patient's heart.
  • Patients with the renal variant phenotype can also benefit from ⁇ -Gal A enzyme replacement therapy.
  • the effect of therapy can be measured by standard tests of renal function, such as 24-hour urine protein level, creatinine clearance, and glomerular filtration rate.
  • the ⁇ -Gal A preparations described herein are substantially free of non- ⁇ -Gal A proteins, such as albumin, non- ⁇ -Gal A proteins produced by the host cell, or proteins isolated from animal tissue or fluid.
  • the preparation preferably comprises part of an aqueous or physiologically compatible fluid suspension or solution.
  • the carrier or vehicle is physiologically compatible so that, in addition to delivery of the desired preparation to the patient, it does not otherwise adversely affect the patient's electrolyte and/or volume balance.
  • Useful solutions for parenteral administration may be prepared by any of the methods well known in the pharmaceutical art (See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES Gennaro, A., ed., Mack Pub., 1990).
  • Non-parenteral formulations such as suppositories and oral formulations
  • the formulation contains an excipient.
  • Pharmaceutically acceptable excipients for ⁇ -Gal A which may be included in the formulation are buffers such as citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer, amino acids, urea, alcohols, ascorbic acid, phospholipids; proteins, such as serum albumin, collagen, and gelatin; salts such as EDTA or EGTA, and sodium chloride; liposomes; polyvinylpyrollidone; sugars, such as dextran, mannitol, sorbitol, and glycerol; propylene glycol and polyethylene glycol (PEG); glycerol; glycine or other amino acids; and lipids.
  • buffers such as citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer, amino acids, urea, alcohols, ascorbic acid, phospholipids
  • proteins such as
  • Preferred excipients include mannitol, sorbitol, glycerol, amino acids, lipids, EDTA, EGTA, sodium chloride, polyethylene glycol, polyvinylpyrollidone, dextran, or combinations of any of these excipients.
  • the formulation further comprises a non-ionic detergent.
  • Preferred non-ionic detergents include Polysorbate 20, Polysorbate 80, Triton X-100TM, Triton X-114TM, Nonidet P-40TM, Octyl ⁇ -glucoside, Octyl ⁇ -glucoside, Brij 35, PluronicTM, Poloxamer 188 (a.k.a. Poloxalkol) and Tween 20TM.
  • the non-ionic detergent comprises Polysorbate 20 or Polysorbate 80.
  • a preferred formulation further comprises phosphate-buffered saline, e.g., at pH 6.
  • Buffer systems for use with ⁇ -Gal A preparations include citrate; acetate; bicarbonate; and phosphate buffers (all available from Sigma). Phosphate buffer is a preferred embodiment.
  • a preferred pH range for ⁇ -Gal A preparations is pH 4.5-7.4.
  • the protein concentration can be 0.1-10 mg/mL.
  • Bulking agents such as glycine, mannitol, albumin, and dextran, can be added to the lyophilization mixture.
  • possible cryoprotectants such as disaccharides, amino acids, and PEG, can be added to the lyophilization mixture. Any of the buffers, excipients, and detergents listed above, can also be added.
  • Formulations for administration may include glycerol and other compositions of high viscosity to help maintain the agent at the desired locus.
  • Biocompatible polymers preferably bioresorbable, biocompatible polymers (including, e.g., hyaluronic acid, collagen, polybutyrate, lactide, and glycolide polymers and lactide/glycolide copolymers) may be useful excipients to control the release of the agent in vivo.
  • Formulations for parenteral administration may include glycocholate for buccal administration, methoxysalicylate for rectal administration, or cutnic acid for vaginal administration.
  • Suppositories for rectal administration may be prepared by mixing an ⁇ -Gal A preparation of the invention with a non-irritating excipient such as cocoa butter or other compositions that are solid at room temperature and liquid at body temperatures.
  • Formulations for inhalation administration may contain lactose or other excipients, or may be aqueous solutions which may contain polyoxyethylene-9-lauryl ether, glycocholate or deoxycocholate.
  • a preferred inhalation aerosol is characterized by having particles of small mass density and large size. Particles with mass densities less than 0.4 gram per cubic centimeter and mean diameters exceeding 5 ⁇ m efficiently deliver inhaled therapeutics into the systemic circulation. Such particles are inspired deep into the lungs and escape the lungs' natural clearance mechanisms until the inhaled particles deliver their therapeutic payload. (Edwards et al., Science 276: 1868-1872 (1997)).
  • ⁇ -Gal A preparations of the present invention can be administered in aerosolized form, for example by using methods of preparation and formulations as described in U.S. Pat. Nos. 5,654,007, 5, 780,014, and 5,814,607, each incorporated herein by reference.
  • Formulation for intranasal administration may include oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • Formulations for topical administration to the skin surface may be prepared by dispersing the ⁇ -Gal A preparation with a dermatological acceptable carrier such as a lotion, cream, ointment, or soap. Particularly useful are carriers capable of forming a film or layer over the skin to localize application and inhibit removal.
  • a dermatological acceptable carrier such as a lotion, cream, ointment, or soap.
  • Particularly useful are carriers capable of forming a film or layer over the skin to localize application and inhibit removal.
  • the ⁇ -Gal A preparation may be dispersed in a liquid tissue adhesive or other substance known to enhance adsorption to a tissue surface.
  • mucosal adhesives and buccal tablets have been described for transmucosal drug delivery, Such as in U.S. Pat. Nos. 4,740,365, 4,764,378, and 5,780,045, each incorporated herein by reference.
  • Hydroxypropylcellulose or fibrinogen/thrombin solutions may also be incorporated.
  • tissue-coating solutions such as pectin-containing formulations may be used.
  • the preparations of the invention may be provided in containers suitable for maintaining sterility, protecting the activity of the active ingredients during proper distribution and storage, and providing convenient and effective accessibility of the preparation for administration to a patient.
  • An injectable formulation of an ⁇ -Gal A preparation might be supplied in a stoppered vial suitable for withdrawal of the contents using a needle and syringe. The vial would be intended for either single use or multiple uses.
  • the preparation can also be supplied as a prefilled syringe.
  • the contents would be supplied in liquid formulation, while in others they would be supplied in a dry or lyophilized state, which in some instances would require reconstitution with a standard or a supplied diluent to a liquid state.
  • the preparation is supplied as a liquid for intravenous administration, it might be provided in a sterile bag or container suitable for connection to an intravenous administration line or catheter.
  • the preparations of the invention are supplied in either liquid or powdered formulations in devices which conveniently administer a predetermined dose of the preparation; examples of such devices include a needle-less injector for either subcutaneous or intramuscular injection, and a metered aerosol delivery device.
  • the preparation may be supplied in a form suitable for sustained release, such as in a patch or dressing to be applied to the skin for transdermal administration, or via erodible devices for transmucosal administration.
  • the preparation might be supplied in a bottle with a removable cover.
  • the containers may be labeled with information such as the type of preparation, the name of the manufacturer or distributor, the indication, the suggested dosage, instructions for propel storage, or instructions for administration.
  • the ⁇ -Gal A preparations described herein may be administered by any route which is compatible with the ⁇ -Gal A preparation.
  • the purified ⁇ -Gal A preparation can be administered to individuals who produce insufficient or defective ⁇ -Gal A protein or who may benefit from ⁇ -Gal A therapy.
  • Therapeutic preparations of the present invention may be provided to an individual by any suitable means, directly (e.g., locally, as by injection, implantation or topical administration to a tissue locus) or systemically (e.g., orally or parenterally).
  • the preferred route of administration is intravenous.
  • Other routes of administration may be oral or parenteral, including subcutaneous, intra-arterial, intraperitoneal, oplithlalmiic, intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral, intradermal, intracranial, intraspinal, intraventricular, intrathecal, intracistemal, intracapsular, intrapulmonary, intranasal, transmucosal, transdermal, or via inhalation.
  • Intrapulmonary delivery methods, apparatus and drug preparation are described, for example, in U.S. Pat. Nos. 5,785, 049, 5,780,019, and 5,775,320, each incorporated herein by reference.
  • a preferred method of intradermal delivery is by iontophoretic delivery via patches; one example of such delivery is taught in U.S. Pat. No. 5,843,015, which is incorporated herein by reference.
  • a particularly useful route of administration is by subcutaneous injection.
  • An ⁇ -Gal A preparation of the present invention is formulated such that the total required dose may be administered in a single injection of one or two milliliters.
  • an ⁇ -Gal A preparation of the present invention may be formulated at a concentration in which the preferred dose is delivered in a volume of one to two milliliters, or the ⁇ -Gal A preparation may be formulated in a lyophilized form, which is reconstituted in water or an appropriate physiologically compatible buffer prior to administration.
  • Subcutaneous injections of ⁇ -Gal A preparations have the advantages of being convenient for the patient, in particular by allowing self-administration, while also resulting in a prolonged plasma half-life as compared to, for example, intravenous administration.
  • a prolongation in plasma half-life results in maintenance of effective plasma ⁇ -Gal A levels over longer time periods, the benefit of which is to increase the exposure of clinically affected tissues to the injected ⁇ -Gal A and, as a result, may increase the uptake of ⁇ -Gal A into such tissues. This allows a more beneficial effect to the patient and/or a reduction in the frequency of administration.
  • a variety of devices designed for patient convenience such as refillable injection pens and needle-less injection devices, may be used with the ⁇ -Gal A preparations of the present invention as discussed herein.
  • Administration may be by periodic injections of a bolus of the preparation, or may be administered by intravenous or intraperitoneal administration from a reservoir which is external (e.g., an IV bag) or internal (e.g., a bioerodable implant, a bioartificial organ, or a population of implanted ⁇ -Gal A production cells). See, e.g., U.S. Pat. Nos. 4,407,957 and 5,798,113, each incorporated herein by reference. Intrapulmonary delivery methods and apparatus are described, for example, in U.S. Pat. Nos. 5,654,007, 5,780, 014, and 5,814,607, each incorporated herein by reference.
  • parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, pump delivery, encapsulated cell delivery, liposomal delivery, needle-delivered injection, needle-less injection, nebulizer, acorosolizer, electroporation, and transdermal patch.
  • Needle-less injector devices are described in U.S. Pat. Nos. 5,879,327; 5520,639; 5,846,233 and 5,704,911, the specifications of which are herein incorporated by reference. Any of the ⁇ -Gal A preparation described above can administered in these methods.
  • the route of administration and the amount of protein delivered can be determined by factors that are well within the ability of skilled artisans to assess. Furthermore, skilled artisans are aware that the route of administration and dosage of a therapeutic protein may be varied for a given patient until a therapeutic dosage level is obtained.
  • a targeting DNA fragment containing an appropriate gene-activating sequence was introduced into host human cell lines by electroporation.
  • One such cell line is HT-1080, a certified cell line available from ATCC (Manassas, Va.).
  • the gene activation plasmid (targeting construct) pGA213C containing such a DNA fragment is shown in FIG. 3A.
  • This plasmid contains sequences designed to activate a portion of the endogenous ⁇ -Gal A locus in the host cell line, and contains sequences encoding the signal peptide, but not human ⁇ -Gal A.
  • the targeting construct also contains expression cassettes for the bacterial neo and mouse dhfr genes. These allow for the selection of stably integrated targeting fragments (via the neo gene) and for subsequent selection of the dhfr gene using step-wise methotrexate (MTX) selection.
  • MTX methotrexate
  • pGA213C contains sequences designed to target chromosomal sequences upstream of the endogenous ⁇ -Gal A locus by homologous recombination. Homologous recombination between the endogenous ⁇ -Gal A locus and the 9.6 kb DNA fragment of pGA213C is shown in FIG. 3B.
  • pGA213C was constructed to delete 962 bp of genomic sequences extending from positions ⁇ 1183 to ⁇ 222 relative to the methionine initiation codon of ⁇ -Gal A, upon homologous recombination of the pGA213C fragment with the X-chromosomal ⁇ -Gal A locus. Transcriptional activation of the ⁇ -Gal A locus occurs through precise targeting of the exogenous regulatory sequences upstream of the ⁇ -Gal A coding region. The resulting GA-GAL locus cause transcription to initiate from the CMV promoter and to proceed through CMV exon 1, the aldolase intron and the seven exons and six introns of the ⁇ -Gal A coding sequence.
  • MTX stepwise methotrexate
  • plasmids Two other expression plasmids, pXAG-16 and pXAG-28, were constructed. These plasmids contain human ⁇ -Gal A cDNA encoding the 398 amino acids of the ⁇ -Gal A enzyme (without the ⁇ -Gal A signal peptide); the human growth hon-none (hGH) signal peptide genomic DNA sequence, which is interrupted by the first intron of the hGH gene; and the untranslated sequence (UTS) of the hGH gene, which contains a signal for polyadenylation.
  • hGH human growth hon-none
  • Plasmid pXAG-16 has the human cytomegalovirus immediate-early (CMV IE) promoter and first intron (flanked by non-coding exon sequences), while pXAG-28 is driven by the collagen I ⁇ 2 promoter and exon 1, and also contains the ⁇ -actin gene's 5′UTS, which contains the first intron of the ⁇ -actin gene.
  • CMV IE human cytomegalovirus immediate-early
  • pXAG-28 is driven by the collagen I ⁇ 2 promoter and exon 1, and also contains the ⁇ -actin gene's 5′UTS, which contains the first intron of the ⁇ -actin gene.
  • ⁇ -Gal A In order to express ⁇ -Gal A in fibroblasts, secondary fibroblasts were cultured and transfected according to published procedures (Selden et al., WO 93/09222). The plasmids pXAG-13, pXAG-16 and pXAG-28 were transfected by electroporation into human foreskin fibroblasts to generate stably transfected clonal cell strains, and the resulting ⁇ -Gal A expression levels were monitored. Secretion of ⁇ -Gal A by normal foreskin fibroblasts is in the range of 2-10 units/10 6 cells/24 hours. In contrast, the transfected fibroblasts displayed mean expression levels as shown in the table below.
  • Cell strains desirable for gene therapy or for use in generation of material for purification of ⁇ -Gal A should display stable growth and expression over several passages. Data from the cell strains which were stably transfected with the ⁇ -Gal A expression construct showed that ⁇ -Gal A expression is stably maintained during serial passage.
  • This example compares the structure of ReplagalTM, an ⁇ -Gal A preparation produced in human cells vs. FabrazymeTM, an ⁇ -Gal A preparation produced in CHO cells. The preparations were compared with respect to isoelectric point, molecular weight, and carbohydrate, phosphorylation and sialylation profile.
  • ReplagalTM and FabrazymeTM were analyzed by denaturing isoelectric focusing (5% gels, pH range 3 to 7, 6 M urea) followed by Western blotting.
  • the preparations were also analyzed by native isoelectric focusing (Novex 5% gels, pH range 3 to 7) following by Coomassie Blue staining.
  • the overall pI range of the 2 preparations were similar, although the relative intensities of the banding patterns were different. This indicates that the glycoforms present in each preparation differ in charge distribution, with FabrazymeTM containing a greater proportion of lower pI (more negatively charged) glycoforms than ReplagalTM.
  • ReplagalTM and FabrazymeTM were analyzed by SDS-PAGE (8-16% polyacrylamide gel, reduced samples) followed by Coomassie Blue staining. The molecular weights of the preparations were similar. However, the lower (approximately 45 k(D) glycoform band of FabrazymeTM is more distinct compared to that of ReplagalTM while ReplagalTM exhibits a broader size distribution.
  • FIG. 8 shows FabrazymeTM (top) and ReplagalTM (bottom) as analyzed by reversed phase HPLC using a C4 reversed phase column (Vydac). Chromatograms obtained at 214 nm are shown. The leading shoulder, corresponding to the lower molecular weight glycoforms, is much more pronounced in FabrazymeTM.
  • FIG. 7 shows charge profiles of the glycans released from ReplagalTM (bottom) and FabrazymeTM (top). Glycans were derivatized with a fluorescent probe and compared by ion exchange chromatography on a GlycoSepTM C column. The results show that ReplagalTM has a higher proportion of neutral and mono-charged glycans, and FabrazymeTM has a higher proportion of tri-charged glycans.
  • Table 1 shows a glycan peak area comparison. Glycans released from ReplagalTM and FabrazymeTM were analyzed using HPAE-PAD as shown in FIG. 4. Integration of peaks was performed to quantify the percentages of the various peak groups. The tabulated data demonstrate a higher proportion of phosphorylated glycans in FabrazymeTM, and a higher proportion of neutral glycans and a higher total proportion of sialylated glycans in ReplagalTM.
  • Table 2 shows charge profile results. Charge profiles of glycans released from ReplagalTM and FabrazymeTM were derivatized and separated as described in FIG. 7. CK-022 and CK-006 are 2 different ReplagalTM preparations, while CK-JL012502 is a preparation of FabrazymeTM. Glycans from each product were assayed in duplicate. As shown in the table, the ReplagalTM preparations have higher proportions of glycans that are neutral or carry 1 charge, while FabrazymeTM contains a higher proportion glycans with 2 or 3 charges.
  • Table 3 shows the desialylated profile results.
  • Charge profiles of glycans released from ReplagalTM and FabrazymeTM were desialylated, followed by derivatization and separation as described in FIG. 7.
  • CK-022 and CK-006 are 2 different ReplagalTM preparations, while CK-JL012502 is a preparation of FabrazymeTM.
  • Glycans from each product were assayed in duplicate.
  • the ReplagalTM preparations have lower proportions of residual charged glycans after desialylation, indicating that FabrazymeTM has a higher proportion of phosphorylated (sialidase-resistant) glycans.
  • Blood samples were collected from male Fabry patients receiving their initial 40 minute infusion of ReplagalTM. Blood samples were processed to either plasma or serum and analyzed for ⁇ -Gal A enzyme activity. Serum concentration profiles were analyzed using a noncompartment model to estimate pharmacokinetic parameters.
  • Liver biopsies were taken 44 hours after dosing from male Fabry patients in the Phase I trial. Tissue samples were processed and analyzed for concentration of administered ⁇ -Gal A as previously described (Schiffman and Brady et al. (2000) Proc. Natl. Acad. Sci. USA 97:365-370). The amount of administered dose recovered in each patient's liver was calculated using the concentration of ⁇ -Gal A in each liver biopsy and each patient's estimated liver weight.
  • ReplagalTM had a biphasic serum elimination profile following a single IV dose in rats, rabbits and monkeys (FIG. 9 illustrates the profile in cynomolgus monkey). C max was proportional to dose for these three animal species (FIG. 10). ReplagalTM also had a biphasic serum elimination profile in Fabry patients following a 40 minute infusion (FIG. 11). C max was also dose proportional in humans (FIG. 12). ReplagalTM was eliminated by 24 hours after dosing in all species. AUC (area under the curve) increased linearly with dose in animals and humans over a dose range of 0.017 to 3.2 ⁇ 10 6 U/kg (FIG. 13). The dose range in U/keg corresponds to a range of 0.007 to 0.2 mg/kg in humans and 0.0625 to 1 mg/kg in animals.
  • the exponent in the scaling equation call be near 1.0 (e.g., blood volume) but varies between 0.6 and 0.8 for drug or protein clearance.
  • Blood samples were collected from male and female Fabry patients receiving their initial 40 minute infusion of ReplagalTM from TKT006 (NIH), TKT007 (UK) and TKT014 (GERMANY). Blood samples were processed to serum (TKT007 and TKT014 samples) or plasma (TKT006 samples) and analyzed for ⁇ -galactosidase A enzyme activity at TKT using an in vitro fluorescence assay. Serum/plasma concentration profiles were analyzed using a noncompartmental model to estimate pharmacokinetic parameters.
  • ReplagalTM had a biphasic serum elimination profile following a single intravenous infusion in both male and female Fabry patients and was eliminated from most patients by 24 hours after dosing (FIG. 15). As expected, C max coincided with the end of the 40 minute infusion period.

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US10/423,225 US20040071686A1 (en) 2002-04-25 2003-04-25 Treatment of alpha-galactosidase A deficiency
US11/403,618 US7833742B2 (en) 2002-04-25 2006-04-13 Treatment of α-galactosidase A deficiency
US12/761,287 US9267166B2 (en) 2002-04-25 2010-04-15 Treatment of α-galactosidase A deficiency
US14/995,540 US9523113B2 (en) 2002-04-25 2016-01-14 Treatment of α-galactosidase A deficiency
US15/350,785 US10245304B2 (en) 2002-04-25 2016-11-14 Treatment of alpha-galactosidase a deficiency
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